Wednesday, November 12, 2008

Q1. what is the source of energy for light production in the firefly ?”

1. ATP2. GTP3. phosho creatine4. NADH

The answer is ATP….

Let me start the discussion with this question …..“DO YOU THINK THAT THE QUESTION IS RELEVANT TO US ?”

If you think this question is irrelevant , then go through the following discussion & then you ll understand why these AIIMSpeople want us to know about the metabolic machinery of this humble organism.

The firefly (Photinus pyralis) contains an enzyme called LUCIFERASE. This enzyme acts on its substrate – luciferin in the presence of ATP and calcium. The reaction produces visible light. This reaction has been exploited by the scientific community for research on

MDR TB is characterized by the presence of resistance to the two drugsIsoniazide …….. due to mutations of genes Kat G & INH ARifampicin……..due the mutations in the gene rpo B gene

To the mycobacteria cultured from the patient`s clinical samples we add adeno virus carrying the FFLUX gene (which codes for luciferase). Within a few minutes the mycobacteria exhibit bioluminescence i.e they emit light. Now add the antibiotic , say INH. If the mycobacteria are sensitive , they soon become non viable & their metabolic machinery is paralysed and hence ATP is depleted. In the absence of ATP , the light production is reduced. In fact the degree of reduction of the bio luminescence can be taken as an indicator of the potency of the drug .If the strains are resistant , there is NO reduction in light production

PYRO SEQUENCING

This is a type of DNA sequencing.Take the template DNA which is to be sequenced. To this we add luciferase, luciferin, calcium , DNA polymerase, ATP sulfurylaseThen we add the deoxy nucleotides (A, C , G , T) one by one and wash them off.For instance let the d-NTP in x position of the template be A. When we add A, G , C there will be no reaction. But when we add T , base pairing occurs , releasing a PYRO PHOSPHATE, which is acted upon by ATP sulfurylase to produce ATP.When there is ATP , luciferase is activated and there is luminescence. So we know that the template contains the d NTP which is complementary to the one that we added.in the same way the entire NA can be sequenced

STUDYING METABOLIC PATHWAYS

Coenorhabditis elegans (a nematode) was the FIRST MULTICELLULAR ORGANISM FOR WHICH THE ENTIRE GENOME WAS SEQUENCED.

An interesting aspect is that its cuticular layer is transparent. We can add the luciferase gene to its genome with the help of adeno or lenti virus. So whenever its metabolic machinery is activated an ATP is produced, light is emitted.Likewise whenever a metabolic poison is given the ‘light is switched off’The degree of bio luminescence is proportional to the amount of ATP present

STUDYING TRANSCRIPTIONAL ACTIVITIES

To study the transcriptional activity of a gene , incorporate the luciferase gene close to the index gene `s promoterWhenever there is transcription, luciferase is also activated and light is emitted

TUMORIGENESIS MODELS

The tumor cells tagged with luciferase gene become luminescent and thereby their growth and susceptibility to chemo therapeutic agents can be studied in vivo within mouse models

OTHERS

Luciferase gene is also used in forensic medicine - detect blood and in studying specific cell lines

Sunday, August 10, 2008

Cholesterol is a lipid found in the cell membranes of all animal tissues, and is transported in the blood plasma of all animals. Cholesterol is also a sterol (a combination steroid and alcohol). Because cholesterol is synthesized by all eukaryotes, trace amounts of cholesterol are also found in membranes of plants and fungi.

The name originates from the Greek chole- (bile) and stereos (solid), and the chemical suffix -ol for an alcohol, as researchers first identified cholesterol in solid form in gallstones by François Poulletier de la Salle in 1769. However, it is only in 1815 that chemist Eugène Chevreul named the compound "cholesterine".

Most of the cholesterol in the body is synthesized by the body and some has dietary origin. Cholesterol is more abundant in tissues which either synthesize more or have more abundant densely-packed membranes, for example, the liver, spinal cord and brain. It plays a central role in many biochemical processes, such as the composition of cell membranes and the synthesis of steroid hormones.

Since cholesterol is insoluble in blood, it is transported in the circulatory system within lipoproteins, complex spherical particles which have an exterior composed mainly of water-soluble proteins; fats and cholesterol are carried internally. There is a large range of lipoproteins within blood, generally called, from larger to smaller size: chylomicrons, very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL), low density lipoprotein (LDL) and high density lipoprotein (HDL). The cholesterol within all the various lipoproteins is identical.

According to the lipid hypothesis, abnormally high cholesterol levels (hypercholesterolemia), or, more correctly, higher concentrations of LDL and lower concentrations of functional HDL are strongly associated with cardiovascular disease because these promote atheroma development in arteries (atherosclerosis). This disease process leads to myocardial infarction (heart attack), stroke and peripheral vascular disease. Since higher blood LDL, especially higher LDL particle concentrations and smaller LDL particle size, contribute to this process more than the cholesterol content of the LDL particles [4], LDL particles are often termed "bad cholesterol" because they have been linked to atheroma formation. On the other hand, high concentrations of functional HDL, which can remove cholesterol from cells and atheroma, offer protection and are sometimes referred to colloquially as "good cholesterol". These balances are mostly genetically determined but can be changed by body build, medications, food choices and other factors.

Thursday, May 29, 2008

check out this page for an animation which excellently depicts the basic principle and mechanism underlying PCR . CLICK HERE

The polymerase chain reaction (PCR) is a technique widely used in molecular biology. It derives its name from one of its key components, a DNA polymerase used to amplify a piece of DNA by in vitro enzymatic replication. As PCR progresses, the DNA thus generated is itself used as template for replication. This sets in motion a chain reaction in which the DNA template is exponentially amplified. With PCR it is possible to amplify a single or few copies of a piece of DNA across several orders of magnitude, generating millions or more copies of the DNA piece. PCR can be extensively modified to perform a wide array of genetic manipulations.

Almost all PCR applications employ a heat-stable DNA polymerase, such as Taq polymerase, an enzyme originally isolated from the bacterium Thermus aquaticus. This DNA polymerase enzymatically assembles a new DNA strand from DNA building blocks, the nucleotides, using single-stranded DNA as template and DNA oligonucleotides (also called DNA primers) required for initiation of DNA synthesis. The vast majority of PCR methods use thermal cycling, i.e., alternately heating and cooling the PCR sample to a defined series of temperature steps. These thermal cycling steps are necessary to physically separate the strands (at high temperatures) in a DNA double helix (DNA melting) used as template during DNA synthesis (at lower temperatures) by the DNA polymerase to selectively amplify the target DNA. The selectivity of PCR results from the use of primers that are complementary to the DNA region targeted for amplification under specific thermal cycling conditions.

Developed in 1983 by Kary Mullis,[1] PCR is now a common and often indispensable technique used in medical and biological research labs for a variety of applications.[2][3] These include DNA cloning for sequencing, DNA-based phylogeny, or functional analysis of genes; the diagnosis of hereditary diseases; the identification of genetic fingerprints (used in forensics and paternity testing); and the detection and diagnosis of infectious diseases. In 1993 Mullis won the Nobel Prize for his work on PCR.[4]

PCR principle

PCR is used to amplify specific regions of a DNA strand (the DNA target). This can be a single gene, a part of a gene, or a non-coding sequence. Most PCR methods typically amplify DNA fragments of up to 10 kilo base pairs (kb), although some techniques allow for amplification of fragments up to 40 kb in size.[5]

A basic PCR set up requires several components and reagents.[6] These components include:

DNA template that contains the DNA region (target) to be amplified.

One or more primers, which are complementary to the DNA regions at the 5' (five prime) and 3' (three prime) ends of the DNA region.

A DNA polymerase such as Taq polymerase or another DNA polymerase with a temperature optimum at around 70°C.

Deoxynucleoside triphosphates (dNTPs; also very commonly and erroneously called deoxynucleotide triphosphates), the building blocks from which the DNA polymerases synthesizes a new DNA strand.

Buffer solution, providing a suitable chemical environment for optimum activity and stability of the DNA polymerase.

Divalent cations, magnesium or manganese ions; generally Mg2+ is used, but Mn2+ can be utilized for PCR-mediated DNA mutagenesis, as higher Mn2+ concentration increases the error rate during DNA synthesis[7]

Monovalent cation potassium ions.

The PCR is commonly carried out in a reaction volume of 15-100 μl in small reaction tubes (0.2-0.5 ml volumes) in a thermal cycler. The thermal cycler heats and cools the reaction tubes to achieve the temperatures required at each step of the reaction (see below). Many modern thermal cyclers make use of the Peltier effect which permits both heating and cooling of the block holding the PCR tubes simply by reversing the electric current. Thin-walled reaction tubes permit favorable thermal conductivity to allow for rapid thermal equilibration. Most thermal cyclers have heated lids to prevent condensation at the top of the reaction tube. Older thermocyclers lacking a heated lid require a layer of oil on top of the reaction mixture or a ball of wax inside the tube.

Procedure

The PCR usually consists of a series of 20 to 35 repeated temperature changes called cycles; each cycle typically consists of 2-3 discrete temperature steps. Most commonly PCR is carried out with cycles that have three temperature steps (Fig. 2). The cycling is often preceded by a single temperature step (called hold) at a high temperature (>90°C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters. These include the enzyme used for DNA synthesis, the concentration of divalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers.[8]

Initialization step: This step consists of heating the reaction to a temperature of 94-96°C (or 98°C if extremely thermostable polymerases are used), which is held for 1-9 minutes. It is only required for DNA polymerases that require heat activation by hot-start PCR.[9]

Denaturation step: This step is the first regular cycling event and consists of heating the reaction to 94-98°C for 20-30 seconds. It causes melting of DNA template and primers by disrupting the hydrogen bonds between complementary bases of the DNA strands, yielding single strands of DNA.

Annealing step: The reaction temperature is lowered to 50-65°C for 20-40 seconds allowing annealing of the primers to the single-stranded DNA template. Typically the annealing temperature is about 3-5 degrees Celsius below the Tm of the primers used. Stable DNA-DNA hydrogen bonds are only formed when the primer sequence very closely matches the template sequence. The polymerase binds to the primer-template hybrid and begins DNA synthesis.

Extension/elongation step: The temperature at this step depends on the DNA polymerase used; Taq polymerase has its optimum activity temperature at 75-80°C,[10][11] and commonly a temperature of 72°C is used with this enzyme. At this step the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding dNTPs that are complementary to the template in 5' to 3' direction, condensing the 5'-phosphate group of the dNTPs with the 3'-hydroxyl group at the end of the nascent (extending) DNA strand. The extension time depends both on the DNA polymerase used and on the length of the DNA fragment to be amplified. As a rule-of-thumb, at its optimum temperature, the DNA polymerase will polymerize a thousand bases per minute. Under optimum conditions, i.e., if there are no limitations due to limiting substrates or reagents, at each extension step, the amount of DNA target is doubled, leading to exponential (geometric) amplification of the specific DNA fragment.

Final elongation: This single step is occasionally performed at a temperature of 70-74°C for 5-15 minutes after the last PCR cycle to ensure that any remaining single-stranded DNA is fully extended.

Final hold: This step at 4-15°C for an indefinite time may be employed for short-term storage of the reaction.

Figure 3: Ethidium bromide-stained PCR products after gel electrophoresis. Two sets of primers were used to amplify a target sequence from three different tissue samples. No amplification is present in sample #1; DNA bands in sample #2 and #3 indicate successful amplification of the target sequence. The gel also shows a positive control, and a DNA ladder containing DNA fragments of defined length for sizing the bands in the experimental PCRs.

To check whether the PCR generated the anticipated DNA fragment (also sometimes referred to as the amplimer or amplicon), agarose gel electrophoresis is employed for size separation of the PCR products. The size(s) of PCR products is determined by comparison with a DNA ladder (a molecular weight marker), which contains DNA fragments of known size, run on the gel alongside the PCR products (see Fig. 3).

PCR stages

The PCR process can be divided into three stages:

Exponential amplification: At every cycle, the amount of product is doubled (assuming 100% reaction efficiency). The reaction is very specific and precise.[citation needed]

Levelling off stage: The reaction slows as the DNA polymerase loses activity and as consumption of reagents such as dNTPs and primers causes them to become limiting.

Plateau: No more product accumulates due to exhaustion of reagents and enzyme.

PCR optimization

In practice, PCR can fail for various reasons, in part due to its sensitivity to contamination causing amplification of spurious DNA products. Because of this, a number of techniques and procedures have been developed for optimizing PCR conditions.[12][13] Contamination with extraneous DNA is addressed with lab protocols and procedures that separate pre-PCR mixtures from potential DNA contaminants.[6] This usually involves spatial separation of PCR-setup areas from areas for analysis or purification of PCR products, and thoroughly cleaning the work surface between reaction setups. Primer-design techniques are important in improving PCR product yield and in avoiding the formation of spurious products, and the usage of alternate buffer components or polymerase enzymes can help with amplification of long or otherwise problematic regions of DNA.

Application of PCR

Isolation of genomic DNA

PCR allows isolation of DNA fragments from genomic DNA by selective amplification of a specific region of DNA. This use of PCR augments many methods, such as generating hybridization probes for Southern or northern hybridization and DNA cloning, which require larger amounts of DNA, representing a specific DNA region. PCR supplies these techniques with high amounts of pure DNA, enabling analysis of DNA samples even from very small amounts of starting material.

Other applications of PCR include DNA sequencing to determine unknown PCR-amplified sequences in which one of the amplification primers may be used in Sanger sequencing, isolation of a DNA sequence to expedite recombinant DNA technologies involving the insertion of a DNA sequence into a plasmid or the genetic material of another organism. Bacterial colonies (E.coli) can be rapidly screened by PCR for correct DNA vector constructs[14]. PCR may also be used for genetic fingerprinting; a forensic technique used to identify a person or organism by comparing experimental DNAs through different PCR-based methods.

Some PCR 'fingerprints' methods have high discriminative power and can be used to identify genetic relationships between individuals, such as parent-child or between siblings, and are used in paternity testing (Fig. 4). This technique may also be used to determine evolutionary relationships among organisms.

Amplification and quantitation of DNA

Because PCR amplifies the regions of DNA that it targets, PCR can be used to analyze extremely small amounts of sample. This is often critical for forensic analysis, when only a trace amount of DNA is available as evidence. PCR may also be used in the analysis of ancient DNA that is thousands of years old. These PCR-based techniques have been successfully used on animals, such as a forty-thousand-year-old mammoth, and also on human DNA, in applications ranging from the analysis of Egyptian mummies to the identification of a Russian Tsar.[15]

Quantitative PCR methods allow the estimation of the amount of a given sequence present in a sample – a technique often applied to quantitatively determine levels of gene expression. Real-time PCR is an established tool for DNA quantification that measures the accumulation of DNA product after each round of PCR amplification.

PCR in diagnosis of diseases

PCR allows early diagnosis of malignant diseases such as leukemia and lymphomas, which is currently the highest developed in cancer research and is already being used routinely.[citation needed] PCR assays can be performed directly on genomic DNA samples to detect translocation-specific malignant cells at a sensitivity which is at least 10,000 fold higher than other methods.[citation needed]

PCR also permits identification of non-cultivatable or slow-growing microorganisms such as mycobacteria, anaerobic bacteria, or viruses from tissue culture assays and animal models. The basis for PCR diagnostic applications in microbiology is the detection of infectious agents and the discrimination of non-pathogenic from pathogenic strains by virtue of specific genes.[citation needed]

Viral DNA can likewise be detected by PCR. The primers used need to be specific to the targeted sequences in the DNA of a virus, and the PCR can be used for diagnostic analyses or DNA sequencing of the viral genome. The high sensitivity of PCR permits virus detection soon after infection and even before the onset of disease. Such early detection may give physicians a significant lead in treatment. The amount of virus ("viral load") in a patient can also be quantified by PCR-based DNA quantitation techniques (see below).

Variations on the basic PCR technique

Allele-specific PCR: This diagnostic or cloning technique is used to identify or utilize single-nucleotide polymorphisms (SNPs) (single base differences in DNA). It requires prior knowledge of a DNA sequence, including differences between alleles, and uses primers whose 3' ends encompass the SNP. PCR amplification under stringent conditions is much less efficient in the presence of a mismatch between template and primer, so successful amplification with an SNP-specific primer signals presence of the specific SNP in a sequence.[16] See SNP genotyping for more information.

Assembly PCR or Polymerase Cycling Assembly (PCA): Assembly PCR is the artificial synthesis of long DNA sequences by performing PCR on a pool of long oligonucleotides with short overlapping segments. The oligonucleotides alternate between sense and antisense directions, and the overlapping segments determine the order of the PCR fragments thereby selectively producing the final long DNA product.[17]

Asymmetric PCR: Asymmetric PCR is used to preferentially amplify one strand of the original DNA more than the other. It finds use in some types of sequencing and hybridization probing where having only one of the two complementary stands is required. PCR is carried out as usual, but with a great excess of the primers for the chosen strand. Due to the slow (arithmetic) amplification later in the reaction after the limiting primer has been used up, extra cycles of PCR are required.[18] A recent modification on this process, known as Linear-After-The-Exponential-PCR (LATE-PCR), uses a limiting primer with a higher melting temperature (Melting temperature|Tm) than the excess primer to maintain reaction efficiency as the limiting primer concentration decreases mid-reaction.[19]

Helicase-dependent amplification: This technique is similar to traditional PCR, but uses a constant temperature rather than cycling through denaturation and annealing/extension cycles. DNA Helicase, an enzyme that unwinds DNA, is used in place of thermal denaturation.[20]

Hot-start PCR: This is a technique that reduces non-specific amplification during the initial set up stages of the PCR. The technique may be performed manually by heating the reaction components to the melting temperature (e.g., 95˚C) before adding the polymerase.[21] Specialized enzyme systems have been developed that inhibit the polymerase's activity at ambient temperature, either by the binding of an antibody[9] or by the presence of covalently bound inhibitors that only dissociate after a high-temperature activation step. Hot-start/cold-finish PCR is achieved with new hybrid polymerases that are inactive at ambient temperature and are instantly activated at elongation temperature.

Intersequence-specific (ISSR) PCR: a PCR method for DNA fingerprinting that amplifies regions between some simple sequence repeats to produce a unique fingerprint of amplified fragment lengths.[22]

Inverse PCR: a method used to allow PCR when only one internal sequence is known. This is especially useful in identifying flanking sequences to various genomic inserts. This involves a series of DNA digestions and self ligation, resulting in known sequences at either end of the unknown sequence.[23]

Ligation-mediated PCR: This method uses small DNA linkers ligated to the DNA of interest and multiple primers annealing to the DNA linkers; it has been used for DNA sequencing, genome walking, and DNA footprinting.[24]

Methylation-specific PCR (MSP): The MSP method was developed by Stephen Baylin and Jim Herman at the JohnsHopkinsSchool of Medicine,[25] and is used to detect methylation of CpG islands in genomic DNA. DNA is first treated with sodium bisulfite, which converts unmethylated cytosine bases to uracil, which is recognized by PCR primers as thymine. Two PCRs are then carried out on the modified DNA, using primer sets identical except at any CpG islands within the primer sequences. At these points, one primer set recognizes DNA with cytosines to amplify methylated DNA, and one set recognizes DNA with uracil or thymine to amplify unmethylated DNA. MSP using qPCR can also be performed to obtain quantitative rather than qualitative information about methylation.

Miniprimer PCR: Miniprimer PCR uses a novel thermostable polymerase (S-Tbr) that can extend from short primers ("smalligos") as short as 9 or 10 nucleotides, instead of the approximately 20 nucleotides required by Taq. This method permits PCR targeting smaller primer binding regions, and is particularly useful to amplify unknown, but conserved, DNA sequences, such as the 16S (or eukaryotic 18S) rRNA gene. 16S rRNA miniprimer PCR was used to characterize a microbial mat community growing in an extreme environment, a hypersaline pond in Puerto Rico. In that study, deeply divergent sequences were discovered with high frequency and included representatives that deﬁned two new division-level taxa, suggesting that miniprimer PCR may reveal new dimensions of microbial diversity.[26] By enlarging the "sequence space" that may be queried by PCR primers, this technique may enable novel PCR strategies that are not possible within the limits of primer design imposed by Taq and other commonly used enzymes.

Multiplex-PCR: The use of multiple, unique primer sets within a single PCR mixture to produce amplicons of varying sizes specific to different DNA sequences. By targeting multiple genes at once, additional information may be gained from a single test run that otherwise would require several times the reagents and more time to perform. Annealing temperatures for each of the primer sets must be optimized to work correctly within a single reaction, and amplicon sizes, i.e., their base pair length, should be different enough to form distinct bands when visualized by gel electrophoresis.

Nested PCR: increases the specificity of DNA amplification, by reducing background due to non-specific amplification of DNA. Two sets of primers are being used in two successive PCRs. In the first reaction, one pair of primers is used to generate DNA products, which besides the intended target, may still consist of non-specifically amplified DNA fragments. The product(s) are then used in a second PCR with a set of primers whose binding sites are completely or partially different from and located 3' of each of the primers used in the first reaction. Nested PCR is often more successful in specifically amplifying long DNA fragments than conventional PCR, but it requires more detailed knowledge of the target sequences.

Overlap-extension PCR: is a genetic engineering technique allowing the construction of a DNA sequence with an alteration inserted beyond the limit of the longest practical primer length.

Quantitative PCR (Q-PCR): is used to measure the quantity of a PCR product (preferably real-time). It is the method of choice to quantitatively measure starting amounts of DNA, cDNA or RNA. Q-PCR is commonly used to determine whether a DNA sequence is present in a sample and the number of its copies in the sample. The method with currently the highest level of accuracy is Quantitative real-time PCR. It is often confusingly known as RT-PCR (Real Time PCR) or RQ-PCR. QRT-PCR or RTQ-PCR are more appropriate contractions. RT-PCR commonly refers to reverse transcription PCR (see below), which is often used in conjunction with Q-PCR. QRT-PCR methods use fluorescent dyes, such as Sybr Green, or fluorophore-containing DNA probes, such as TaqMan, to measure the amount of amplified product in real time.

RT-PCR: (Reverse Transcription PCR) is a method used to amplify, isolate or identify a known sequence from a cellular or tissue RNA. The PCR is preceded by a reaction using reverse transcriptase to convert RNA to cDNA. RT-PCR is widely used in expression profiling, to determine the expression of a gene or to identify the sequence of an RNA transcript, including transcription start and termination sites and, if the genomic DNA sequence of a gene is known, to map the location of exons and introns in the gene. The 5' end of a gene (corresponding to the transcription start site) is typically identified by an RT-PCR method, named RACE-PCR, short for Rapid Amplification of cDNA Ends.

Solid Phase PCR: encompasses multiple meanings, including Polony Amplification (where PCR colonies are derived in a gel matrix, for example), 'Bridge PCR' (the only primers present are covalently linked to solid support surface), conventional Solid Phase PCR (where Asymmetric PCR is applied in the presence of solid support bearing primer with sequence matching one of the aqueous primers) and Enhanced Solid Phase PCR[27] (where conventional Solid Phase PCR can be improved by employing high Tm solid support primer with application of a thermal 'step' to favour solid support priming).

TAIL-PCR: Thermal asymmetric interlaced PCR is used to isolate unknown sequence flanking a known sequence. Within the known sequence TAIL-PCR uses a nested pair of primers with differing annealing temperatures; a degenerate primer is used to amplify in the other direction from the unknown sequence.[28]

Touchdown PCR: a variant of PCR that aims to reduce nonspecific background by gradually lowering the annealing temperature as PCR cycling progresses. The annealing temperature at the initial cycles is usually a few degrees (3-5˚C) above the Tm of the primers used, while at the later cycles, it is a few degrees (3-5˚C) below the primer Tm. The higher temperatures give greater specificity for primer binding, and the lower temperatures permit more efficient amplification from the specific products formed during the initial cycles.[29]

PAN-AC: This method uses isothermal conditions for amplification, and may be used in living cells.[30][31]

Universal Fast Walking: this method allows genome walking and genetic fingerprinting using a more specific 'two-sided' PCR than conventional 'one-sided' approaches (using only one gene-specific primer and one general primer - which can lead to artefactual 'noise') [32] by virtue of a mechanism involving lariat structure formation. Streamlined derivatives of UFW are LaNe RAGE (lariat-dependent nested PCR for rapid amplification of genomic DNA ends) [33], 5'RACE LaNe[34] and 3'RACE LaNe[35].

History

A 1971 paper in the Journal of Molecular Biology by Kleppe and co-workers first described a method using an enzymatic assay to replicate a short DNA template with primers in vitro. However, this early manifestation of the basic PCR principle did not receive much attention, and the invention of the polymerase chain reaction in 1983 is generally credited to Kary Mullis.[37]

At the core of the PCR method is the use of a suitable DNA polymerase able to withstand the high temperatures of >90°C (>195°F) required for separation of the two DNA strands in the DNA double helix after each replication cycle. The DNA polymerases initially employed for in vitro experiments presaging PCR were unable to withstand these high temperatures. So the early procedures for DNA replication were very inefficient, time consuming, and required large amounts of DNA polymerase and continual handling throughout the process.

A 1976 discovery of Taq polymerase a DNA polymerase purified from the thermophilic bacterium, Thermus aquaticus, which naturally occurs in hot (50 to 80 °C (120 to 175 °F)) environments paved the way for dramatic improvements of the PCR method. The DNA polymerase isolated from T. aquaticus is stable at high temperatures remaining active even after DNA denaturation,thus obviating the need to add new DNA polymerase after each cycle. This allowed an automated thermocycler-based process for DNA amplification.

At the time he developed PCR in 1983, Mullis was working in Emeryville, California for Cetus Corporation, one of the first biotechnology companies. There, he was responsible for synthesizing short chains of DNA. Mullis has written that he conceived of PCR while cruising along the Pacific Coast Highway one night in his car. He was playing in his mind with a new way of analyzing changes (mutations) in DNA when he realized that he had instead invented a method of amplifying any DNA region through repeated cycles of duplication driven by DNA polymerase.

In Scientific American, Mullis summarized the procedure: "Beginning with a single molecule of the genetic material DNA, the PCR can generate 100 billion similar molecules in an afternoon. The reaction is easy to execute. It requires no more than a test tube, a few simple reagents, and a source of heat." He was awarded the Nobel Prize in Chemistry in 1993 for his invention,seven years after he and his colleagues at Cetus first put his proposal to practice. However, some controversies have remained about the intellectual and practical contributions of other scientists to Mullis' work, and whether he had been the sole inventor of the PCR principle.

Patent wars

The PCR technique was patented by Cetus Corporation, where Mullis worked when he invented the technique in 1983. The Taq polymerase enzyme was also covered by patents. There have been several high-profile lawsuits related to the technique, including an unsuccessful lawsuit brought by DuPont. The pharmaceutical company Hoffmann-La Roche purchased the rights to the patents in 1992 and currently holds those that are still protected.

A related patent battle over the Taq polymerase enzyme is still ongoing in several jurisdictions around the world between Roche and Promega. The legal arguments have extended beyond the life of the original PCR and Taq polymerase patents, which expired on March 28, 2005

DNA Polymerase 1 has both proof reading and excision repair activity , that is it has both 3' - 5' exonuclease activity and 5' - 3' exonuclease activity respectively. where as the DNA polymerase 2 and 3 have only proof reading activity and no excision repair activity, that is they have 3' - 5' exonuclease activity and no 5' - 3' exonuclease activity.

all the three DNA polymerases have only 5' -3' polymerase activity only.

Thursday, March 6, 2008

Vitamin B6

Vitamin B6 is a water-soluble vitamin that was first isolated in the 1930s. There are three traditionally considerd forms of vitamin B6: pyridoxal (PL), pyridoxine (PN), pyridoxamine (PM). The phosphate ester derivative pyridoxal 5'-phosphate (PLP) is the principal coenzyme form and has the most importance in human metabolism .

Function

Vitamin B6 must be obtained from the diet because humans cannot synthesize it. PLP plays a vital role in the function of approximately 100 enzymes that catalyze essential chemical reactions in the human body . For example, PLP functions as a coenzyme for glycogen phosphorylase, an enzyme that catalyzes the release of glucose from stored glycogen. Much of the PLP in the human body is found in muscle bound to glycogen phosphorylase. PLP is also a coenzyme for reactions used to generate glucose from amino acids, a process known as gluconeogenesis .

Nervous system function

In the brain, the synthesis of the neurotransmitter, serotonin, from the amino acid, tryptophan, is catalyzed by a PLP-dependent enzyme. Other neurotransmitters, such as dopamine, norepinephrine and gamma-aminobutyric acid (GABA), are also synthesized using PLP-dependent enzymes .

Red blood cell formation and function

PLP functions as a coenzyme in the synthesis of heme, an iron-containing component of hemoglobin. Hemoglobin is found in red blood cells and is critical to their ability to transport oxygen throughout the body. Both PL and PLP are able to bind to the hemoglobin molecule and affect its ability to pick up and release oxygen. However, the impact of this on normal oxygen delivery to tissues is not known .

Niacin formation

The human requirement for another B vitamin, niacin, can be met in part by the conversion of the essential amino acid, tryptophan, to niacin, as well as through dietary intake. PLP is a coenzyme for a critical reaction in the synthesis of niacin from tryptophan; thus, adequate vitamin B6 decreases the requirement for dietary niacin.

Hormone function

Steroid hormones, such as estrogen and testosterone, exert their effects in the body by binding to steroid hormone receptors in the nucleus of the cell and altering gene transcription. PLP binds to steroid receptors in a manner that inhibits the binding of steroid hormones, thus decreasing their effects. The binding of PLP to steroid receptors for estrogen, progesterone, testosterone, and other steroid hormones suggests that the vitamin B6 status of an individual may have implications for diseases affected by steroid hormones, including breast cancer and prostate cancers.

Nucleic acid synthesis

PLP serves as a coenzyme for a key enzyme involved in the mobilization of single-carbon functional groups (one-carbon metabolism). Such reactions are involved in the synthesis of nucleic acids. The effect of vitamin B6 deficiency on the function of the immune system may be partly related to the role of PLP in one-carbon metabolism (see Disease Prevention).

Deficiency

Severe deficiency of vitamin B6 is uncommon. Alcoholics are thought to be most at risk of vitamin B6 deficiency due to low dietary intakes and impaired metabolism of the vitamin. In the early 1950s, seizures were observed in infants as a result of severe vitamin B6 deficiency caused by an error in the manufacture of infant formula. Abnormal electroencephalogram (EEG) patterns have been noted in some studies of vitamin B6 deficiency. Other neurologic symptoms noted in severe vitamin B6 deficiency include irritability, depression, and confusion; additional symptoms include inflammation of the tongue, sores or ulcers of the mouth, and ulcers of the skin at the corners of the mouth.

The Recommended Dietary Allowance (RDA)

Because vitamin B6 is involved in many aspects of metabolism, several factors are likely to effect an individual's requirement for vitamin B6. Of those factors, protein intake has been the most studied. Increased dietary protein results in an increased requirement for vitamin B6, probably because PLP is a coenzyme for many enzymes involved in amino acid metabolism . Unlike previous recommendations, the Food and Nutrition Board (FNB) of the Institute of Medicine did not express the most recent RDA for vitamin B6 in terms of protein intake, although the relationship was considered in setting the RDA . The current RDA was revised by the Food and Nutrition Board (FNB) in 1998 and is presented in the table below.

Recommended Dietary Allowance (RDA) for Vitamin B6

Life Stage

Age

Males (mg/day)

Females (mg/day)

Infants

0-6 months

0.1 (AI)

0.1 (AI)

Infants

7-12 months

0.3 (AI)

0.3 (AI)

Children

1-3 years

0.5

0.5

Children

4-8 years

0.6

0.6

Children

9-13 years

1.0

1.0

Adolescents

14-18 years

1.3

1.2

Adults

19-50 years

1.3

1.3

Adults

51 years and older

1.7

1.5

Pregnancy

all ages

-

1.9

Breast-feeding

all ages

-

2.0

Disease Prevention

Homocysteine and cardiovascular disease

Even moderately elevated levels of homocysteine in the blood have been associated with increased risk for cardiovascular disease, including heart disease and stroke . During protein digestion, amino acids, including methionine, are released. Homocysteine is an intermediate in the metabolism of methionine. Healthy individuals utilize two different pathways to metabolize homocysteine. One pathway converts homocysteine back to methionine and is dependent on folic acid and vitamin B12. The other pathway converts homocysteine to the amino acid cysteine and requires two vitamin B6(PLP)-dependent enzymes. Thus, the amount of homocysteine in the blood is regulated by at least three vitamins: folic acid, vitamin B12, and vitamin B6 (diagram). Several large observational studies have demonstrated an association between low vitamin B6 intake or status with increased blood homocysteine levels and increased risk of cardiovascular diseases. A large prospective study found the risk of heart disease in women who consumed, on average, 4.6 mg of vitamin B6 daily was only 67% of the risk in women who consumed an average of 1.1 mg daily . Another large prospective study found higher plasma levels of PLP were associated with a decreased risk of cardiovascular disease independent of homocysteine levels . Further, several studies have reported that low plasma PLP status is a risk factor for coronary artery disease . In contrast to folic acid supplementation, studies supplementing individuals with only vitamin B6 have not resulted in significant decreases in basal (fasting) levels of homocysteine. However, one study found that vitamin B6 supplementation was effective in lowering blood homocysteine levels after an oral dose of methionine (methionine load test) was given , suggesting vitamin B6 may play a role in the metabolism of homocysteine after meals.

Immune function

Low vitamin B6 intake and nutritional status have been associated with impaired immune function, especially in the elderly. Decreased production of immune system cells known as lymphocytes, as well as decreased production of an important immune system protein called interleukin-2, have been reported in vitamin B6 deficient individuals . Restoration of vitamin B6 status has resulted in normalization of lymphocyte proliferation and interleukin-2 production, suggesting that adequate vitamin B6 intake is important for optimal immune system function in older individuals . However, one study found that the amount of vitamin B6 required to reverse these immune system impairments in the elderly was 2.9 mg/day for men and 1.9 mg/day for women; these vitamin B6 requirements are higher than the current RDA.

Cognitive function

A few studies have associated cognitive decline in the elderly or Alzheimer's disease with inadequate nutritional status of folic acid, vitamin B12, and vitamin B6 and thus, elevated levels of homocysteine . One observational study found that higher plasma vitamin B6 levels were associated with better performance on two measures of memory, but plasma vitamin B6 levels were unrelated to performance on 18 other cognitive tests . Similarly, a double-blind, placebo-controlled study in 38 healthy elderly men found that vitamin B6 supplementation improved memory but had no effect on mood or mental performance (19). Further, a placebo-controlled trial in 211 healthy younger, middle-aged, and older women found that vitamin B6 supplementation (75 mg/day) for five weeks improved memory performance in some age groups but had no effect on mood (20). Recently, a systematic review of randomized trials concluded that there is inadequate evidence that supplementation with vitamin B6, vitamin B12, or folic acid improves cognition in those with normal or impaired cognitive function . Because of mixed findings, it is presently unclear whether supplementation with B vitamins might blunt cognitive decline in the elderly. Further, it is not known if marginal B vitamin deficiencies, which are relatively common in the elderly, even contribute to age-associated declines in cognitive function, or whether both result from processes associated with aging and/or disease.

Kidney stones

A large prospective study examined the relationship between vitamin B6 intake and the occurrence of symptomatic kidney stones in women. A group of more than 85,000 women without a prior history of kidney stones were followed over 14 years and those who consumed 40 mg or more of vitamin B6 daily had only two thirds the risk of developing kidney stones compared with those who consumed 3 mg or less . However, in a group of more than 45,000 men followed over six years, no association was found between vitamin B6 intake and the occurrence of kidney stones. Limited data have shown that supplementation of vitamin B6 at levels higher than the tolerable upper intake level (100 mg/day) decreases elevated urinary oxalate levels, an important determinant of calcium oxalate kidney stone formation in some individuals. However, it is less clear that supplementation actually resulted in decreased formation of calcium oxalate kidney stones. Presently, the relationship between vitamin B6 intake and the risk of developing kidney stones requires further study before any recommendations can be made.

Disease Treatment

Vitamin B6 supplements at pharmacologic doses (i.e., doses much larger than those needed to prevent deficiency) have been used in an attempt to treat a wide variety of conditions, some of which are discussed below. In general, well designed, placebo-controlled studies have shown little evidence that large supplemental doses of vitamin B6 are beneficial .

Side effects of oral contraceptives

Because vitamin B6 is required for the metabolism of the amino acid tryptophan, the tryptophan load test (an assay of tryptophan metabolites after an oral dose of tryptophan) was used as a functional assessment of vitamin B6 status. Abnormal tryptophan load tests in women taking high-dose oral contraceptives in the 1960s and 1970s suggested that these women were vitamin B6 deficient. Abnormal results in the tryptophan load test led a number of clinicians to prescribe high doses (100-150 mg/day) of vitamin B6 to women in order to relieve depression and other side effects sometimes experienced with oral contraceptives. However, most other indices of vitamin B6 status were normal in women on high-dose oral contraceptives, and it is unlikely that the abnormality in tryptophan metabolism was due to vitamin B6 deficiency . A more recent placebo-controlled study in women on the lower dose oral contraceptives, which are commonly prescribed today, found that doses up to 150 mg/day of vitamin B6 (pyridoxine) had no benefit in preventing side effects, such as nausea, vomiting, dizziness, depression, and irritability .

Premenstrual syndrome (PMS)

The use of vitamin B6 to relieve the side effects of high-dose oral contraceptives led to the use of vitamin B6 in the treatment of premenstrual syndrome (PMS). PMS refers to a cluster of symptoms, including but not limited to fatigue, irritability, moodiness/depression, fluid retention, and breast tenderness, that begin sometime after ovulation (mid-cycle) and subside with the onset of menstruation (the monthly period). A review of 12 placebo-controlled double-blind trials on vitamin B6 use for PMS treatment concluded that evidence for a beneficial effect was weak . A more recent review of 25 studies suggested that supplemental vitamin B6, up to 100 mg/day, may be of value to treat PMS; however, only limited conclusions could be drawn because most of the studies were of poor quality .

Depression

Because a key enzyme in the synthesis of the neurotransmitters serotonin and norepinephrine is PLP-dependent, it has been suggested that vitamin B6 deficiency may lead to depression. However, clinical trials have not provided convincing evidence that vitamin B6 supplementation is an effective treatment for depression, though vitamin B6 may have therapeutic efficacy in premenopausal women.

Morning sickness (nausea and vomiting in pregnancy)

Vitamin B6 has been used since the 1940s to treat nausea during pregnancy. Vitamin B6 was included in the medication Bendectin, which was prescribed for the treatment of morning sickness and later withdrawn from the market due to unproven concerns that it increased the risk of birth defects. Vitamin B6 itself is considered safe during pregnancy and has been used in pregnant women without any evidence of fetal harm . The results of two double-blind, placebo-controlled trials that used 25 mg of pyridoxine every eight hours for three days or 10 mg of pyridoxine every eight hours for five days suggest that vitamin B6 may be beneficial in alleviating morning sickness. Each study found a slight but significant reduction in nausea or vomiting in pregnant women. A recent systematic review of placebo-controlled trials on nausea during early pregnancy found vitamin B6 to be somewhat effective . However, it should be noted that morning sickness also resolves without any treatment, making it difficult to perform well-controlled trials.

Carpal tunnel syndrome

Carpal tunnel syndrome causes numbness, pain, and weakness of the hand and fingers due to compression of the median nerve at the wrist. It may result from repetitive stress injury of the wrist or from soft tissue swelling, which sometimes occurs with pregnancy or hypothyroidism. Several early studies by the same investigator suggested that vitamin B6 status was low in individuals with carpal tunnel syndrome and that supplementation with 100-200 mg/day over several months was beneficial . A recent study in men not taking vitamin supplements found that decreased blood levels of PLP were associated with increased pain, tingling, and nocturnal wakening, all symptoms of carpal tunnel syndrome . Studies using electrophysiological measurements of median nerve conduction have largely failed to find an association between vitamin B6 deficiency and carpal tunnel syndrome. While a few trials have noted some symptomatic relief with vitamin B6 supplementation, double-blind, placebo-controlled trials have not generally found vitamin B6 to be effective in treating carpal tunnel syndrome.

Sources

Food sources

Surveys in the U.S. have shown that dietary intake of vitamin B6 averages about 2 mg/day for men and 1.5 mg/day for women. A survey of elderly individuals found that men and women over 60 years old consumed about 1.2 mg/day and 1.0 mg/day, respectively; both intakes are lower than the current RDA. Certain plant foods contain a unique form of vitamin B6 called pyridoxine glucoside; this form of vitamin B6 appears to be only about half as bioavailable as vitamin B6 from other food sources or supplements. Vitamin B6 in a mixed diet has been found to be approximately 75% bioavailable . In most cases, including foods in the diet that are rich in vitamin B6 should supply enough to prevent deficiency. However, those who follow a very restricted vegetarian diet might need to increase their vitamin B6 intake by eating foods fortified with vitamin B6 or by taking a supplement. Some foods that are relatively rich in vitamin B6 and their vitamin B6 content in milligrams (mg) are listed in the table below. For more information on the nutrient content of specific foods, search the USDA food composition database.

Food

Serving

Vitamin B6 (mg)

Fortified cereal

1 cup

0.5-2.5

Banana

1 medium

0.43

Salmon, wild, cooked

3 ounces*

0.48

Turkey, without skin, cooked

3 ounces

0.39

Chicken, light meat without skin, cooked

3 ounces

0.51

Potato, Russet, baked, with skin

1 medium

0.70

Spinach, cooked

1 cup

0.44

Hazelnuts, dry roasted

1 ounce

0.18

Vegetable juice cocktail

6 ounces

0.26

*A 3-ounce serving of meat or fish is about the size of a deck of cards.

Supplements

Vitamin B6 is available as pyridoxine hydrochloride in multivitamin, vitamin B-complex, and vitamin B6 supplements .

Safety

Toxicity

Because adverse effects have only been documented from vitamin B6 supplements and never from food sources, safety concerning only the supplemental form of vitamin B6 (pyridoxine) is discussed. Although vitamin B6 is a water-soluble vitamin and is excreted in the urine, long-term supplementation with very high doses of pyridoxine may result in painful neurological symptoms known as sensory neuropathy. Symptoms include pain and numbness of the extremities and in severe cases, difficulty walking. Sensory neuropathy typically develops at doses of pyridoxine in excess of 1,000 mg per day. However, there have been a few case reports of individuals who developed sensory neuropathies at doses of less than 500 mg daily over a period of months. Yet, none of the studies in which an objective neurological examination was performed reported evidence of sensory nerve damage at intakes below 200 mg pyridoxine daily . To prevent sensory neuropathy in virtually all individuals, the Food and Nutrition Board of the Institute of Medicine set the tolerable upper intake level (UL) for pyridoxine at 100 mg/day for adults (see table below) . Because placebo-controlled studies have generally failed to show therapeutic benefits of high doses of pyridoxine, there is little reason to exceed the UL of 100 mg/day.

Tolerable Upper Intake Level (UL) for Vitamin B6

Age Group

UL (mg/day)

Infants 0-12 months

Not possible to establish*

Children 1-3 years

30

Children 4-8 years

40

Children 9-13 years

60

Adolescents 14-18 years

80

Adults 19 years and older

100

*Source of intake should be from food and formula only.

Drug interactions

Certain medications interfere with the metabolism of vitamin B6; therefore, some individuals may be vulnerable to a vitamin B6 deficiency if supplemental vitamin B6 is not taken. Anti-tuberculosis medications, including isoniazid and cycloserine, the metal chelator penicillamine, and antiparkinsonian drugs including L-dopa, all form complexes with vitamin B6 and thus create a functional deficiency. Additionally, the efficacy of other medications may be altered by high doses of vitamin B6. For instance, high doses of vitamin B6 have been found to decrease the efficacy of two anticonvulsants, phenobarbital and phenytoin, as well as L-dopa.

Linus Pauling Institute Recommendation

Metabolic studies suggest that young women require 0.02 mg of vitamin B6 per gram of protein consumed daily. Using the upper boundary for acceptable levels of protein intake for women (100 grams/day), the daily vitamin B6 requirement for young women would be calculated at 2.0 mg daily. Older adults may also require at least 2.0 mg/day. For these reasons, the Linus Pauling Institute recommends that all adults consume at least 2.0 mg of vitamin B6 daily. Following the Linus Pauling Institute recommendation to take a daily multivitamin-mineral supplement containing 100% of the Daily Value for vitamin B6 will ensure an intake of at least 2.0 mg/day of vitamin B6. Although a vitamin B6 intake of 2.0 mg daily is slightly higher than the most recent RDA, it is 50 times less than the tolerable upper intake level (UL) set by the Food and Nutrition Board (see Safety).

Older adults (65 years and older)

Metabolic studies have indicated that the requirement for vitamin B6 in older adults is approximately 2.0 mg daily ; this requirement could be even higher if the effect of marginally deficient vitamin B6 intakes on immune function and homocysteine levels are clarified. Despite evidence that the requirement for vitamin B6 may be slightly higher in older adults, several surveys have found that over half of individuals over age 60 consume less than the current RDA (1.7 mg/day for men and 1.5 mg/day for women). For these reasons, the Linus Pauling Institute recommends that older adults take a multivitamin/multimineral supplement, which generally provides at least 2.0 mg of vitamin B6 daily.

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